Proteomics

Mass spectrometers operated at the Institute of Physiology II (Prof. Dr. Bernd Fakler):

• QExactive HF-X (Thermo)
• LTQ-Orbitrap Elite (Thermo)
• LTQ-Orbitrap XL (Thermo)

All instruments are equipped with nano-HPLC-Systems and Nano-spray ionization units.

 

The central goal of our research is to understand the molecular principles underlying fast signaling at and across the plasma membrane. Speed and functional specificity of this signaling are primarily encoded by protein-protein interactions that give rise to protein (super)complexes, networks and (sub)organellar compartments.

To characterize these higher-order assemblies in native cells and tissues we have established three functional proteomic platforms

• Multi-epitope and knock-out controlled affinity-purifications combined with quantitative high-resolution mass spectrometry (meAP-MS)
• Complexome-profiling via cryo-slicing and BN-PAGE-based mass spectrometry (csBN-MS) and
• Organellar proteomics built on multi-dimensional organellar (sub)fractionation (by e.g. multi-step gradient centrifugations) coupled to LC-MS and multi-dimensional cluster analyses.

Our MS-based analyses use either label-free quantification of protein amounts on in-house developed procedures and algorithms, or quantification based on stable isotope labelling (e.g. SILAC). In addition, we pursue exploratory projects on the identification of post-translational modifications of and small molecule ligand binding to selected target proteins. Our in-house MS facility operates with three hybrid tandem mass spectrometers (Orbitrap XL, Orbitrap Elite, QExactive HF-X, each coupled to a nano-HPLC system) covering the specific requirements of our projects with respect to cycling/sequencing speed, sensitivity and mass accuracy.